(Diptera: Cecidomyiidae) infesting Tecticornia ... - Wiley Online Library

Australian Journal of Entomology (2011) 50, 393–404

Taxonomy of two new species of gall midge (Diptera: Cecidomyiidae) infesting Tecticornia arbuscula (Salicornioideae: Chenopodiaceae) in Australian saltmarshes aen_833

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Anneke A Veenstra,1* Agnes Michalczyk1 and Peter Kolesik2 1

Centre for Cell & Molecular Biology, School of Life and Environmental Sciences, Deakin University – Melbourne Campus, 221 Burwood Highway, Burwood, Vic. 3125, Australia. 2 Bionomics Limited, 31 Dalgleish Street, Thebarton, SA 5031, Australia.

Abstract

Two new species of gall midge associated with two leaf galls on the branched, perennial shrub Tecticornia arbuscula are described from saltmarshes in south-eastern Australia. The infestations caused by the new species hinder the growth of T. arbuscula which can impact on the critically endangered Orange Bellied Parrot (Neophema chrysogaster): T. arbuscula provides perching and roosting sites and the seeds are the major food source for this bird. Asphondylia tecticorniae sp. n. Veenstra & Kolesik transforms leaf segments into single-chambered, spherical galls, whereas Asphondylia peelei sp. n. Veenstra & Kolesik produces a multi-chambered, asymmetrical gall on leaves of the same plant. Both galls have fungal mycelium lining the inner surface of the larval chamber where it is presumably grazed on by the larva. Descriptions of the larvae, pupae, males, females and geographical distribution of the two gall midges in south-eastern Australia are given. Differences in the level of parasitoid infestation of four Asphondylia species feeding on Australian Chenopodiaceae in relation to putative oviposition sites on the host plants are explored.

Key words

Asphondylia, Botrysphaeria, insect taxonomy, Platygastridae, Sclerostegia, Tecticornia.

I NTRODUCT IO N The Shrubby Glasswort, Tecticornia arbuscula (syn. Sclerostegia arbuscula) is an up to 2-m-tall perennial shrub, common in coastal and near-coastal saltmarshes of southern New South Wales, Victoria, Tasmania, South Australia and south-east Western Australia (Wilson 1980, 1984). Similar to many other saltmarsh chenopods it has leaves modified into succulent articles and simple, inconspicuous axial flowers. Saltmarsh communities are more floristically diverse than mangroves (Rogers et al. 2005) and more susceptible to damage by trampling, recreational vehicles and changes in tidal regime (Laegdsgaard et al. 2009). In south-eastern Australia, mangrove encroachment of saltmarshes has also contributed to saltmarsh decline (Saintilan & Williams 1999). The incursion of mangrove plant species into saltmarshes in response to sea level rise can be prevented by the presence of species like T. arbuscula where the plants are of similar size and stature (Fig. 1) to the White Mangrove (Avicennia marina) (Laegdsgaard et al. 2009). Dense stands of T. arbuscula are thought to play an inhibitive role by either shading the A. marina seedlings or collecting sediments and building up the marsh surface to exclude mangroves (Rogers et al. 2005). Similar to seeds of Sarcocornia quinqueflora, the developing seeds of T. arbuscula are a major food source of the *[email protected] © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

critically endangered Orange Bellied Parrot (Neophema chrysogaster), which also uses the T. arbuscula branches for perching and roosting (Loyn et al. 1986; OBPRT 1998). This paper describes two new species of gall midge from the genus Asphondylia. It includes geographical distribution of the two gall midges in south-eastern Australia based on the presence of galls on T. arbuscula specimens lodged at six herbaria. Differences in the level of parasitoid infestation of four Asphondylia species feeding on Australian Chenopodiaceae in relation to putative oviposition sites on the host plants are also explored.

MAT ERIAL S AN D MET H ODS Specimen collection and preparation Galls were collected from T. arbuscula growing in saltmarsh within Yaringa Marine National Park, Tyabb, Victoria (38°15′S, 145°14′E), during spring and summer 2008. A small number of galls were cut open and the larvae or pupae preserved in 70% ethanol. The remaining galls were retained in plastic bags to rear adults. Pupation took place within the galls. Plastic bags were examined daily and all emerged adults were preserved in 70% ethanol. Canada balsam or distyrene, tricresyl phosphate and xylene (DPX) mounts of adults, larvae and pupae were prepared according to the technique outlined by doi:10.1111/j.1440-6055.2011.00833.x

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A A Veenstra et al. for amplification were COIS and COIA (Yukawa et al. 2003). DNA sequencing was carried out and the sequences deposited in GenBank. Alignment of sequences and phylogenic analyses were performed as described in Kolesik et al. (2005). Pairwise analysis of 23 sequences were conducted in MEGA4 (Tamura et al. 2007) with a total of 294 positions in the final dataset. Dasineura rubiformis Kolesik (GenBank accession numbers AY278712 and AY278738 described in Kolesik et al. (2005)) was the designated outgroup in phylogenetic analysis.

Geographical distribution Fig. 1. Saltmarsh in Yaringa Marine National Park, Western Port Bay, Victoria. Avicennia marina (White Mangrove) in the foreground and Tecticornia arbuscula (Shrubby Glasswort) behind. Dense stands of T. arbuscula are thought to minimise mangrove incursion into saltmarsh. Stands of Sarcocornia quinqueflora (Beaded Glasswort) and Melaleuca ericifolia (Swamp Paperbark) are present in the landward region of the salt marsh.

Kolesik et al. (2005) and studied microscopically. Measurements refer to the type series. The mounted type series are deposited in the South Australia Museum (SAMA) and the Australian Insect Collection (ANIC) in Canberra. In 2006 and 2008 a number of pupae were collected from the type locality, removed from galls of Asphondylia tecticorniae sp. n. and A. peelei sp. n. immediately after collection and stored in plastic bags at -20°C prior to DNA extraction and subsequent sequencing. Additionally, DNA was extracted from pupae or adults of Asphondylia ericiformis Kolesik (specimens collected with the holotype from Lyndhurst, SA (30°17′S, 138°21′E) by P.K. in 1996 and stored in 70% ethanol at room temperature at SAMA) and Asphondylia inflata Kolesik (specimens collected with the holotype from Port Adelaide, SA (34°50′S, 138°30′E) by P.K. in 1996, and stored in 70% ethanol at room temperature at SAMA).

Gall histology Galls of A. tecticorniae sp. n. Veenstra & Kolesik were collected and preserved in either neutral buffered formalin or 4% paraformaldehyde dissolved in phosphate buffer, prior to dehydration, infiltration and embedding in paraffin wax, sectioning with a microtome (O’Brien & McCully 1981), staining with Mallory’s trichrome stain, and histological examination with a Nikon Coolscope Digital microscope Model II.

DNA extraction and analysis Individual pupae and or larvae were processed for DNA extraction following the method described in Kolesik and Veenstra-Quah (2008). Mitochondrial cytochrome c oxidase subunit I (COI) was used, as it is recommended as a standard gene for ‘DNA barcoding’ by Consortium for the Barcode of Life (http://www.barcodeoflife.org), and the primers used © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

The geographic distribution of both Asphondylia Loew species (A. tecticorniae sp. n. Veenstra & Kolesik or A. peelei sp. n. Veenstra & Kolesik) (Fig. 5) was assessed by investigating the presence of galls on plant specimens of T. arbuscula lodged in the National Herbarium of Victoria (MEL) (n = 55), Tasmanian Herbarium (HO) (n = 14), Western Australian Herbarium (PERTH) (n = 30), National Herbarium Canberra (CANB) (n = 12), National Herbarium of New South Wales (NSW) (n = 6) and the State Herbarium of South Australia, Adelaide (AD) (n = 101) (Table 1). It was not possible to determine which of the two new Asphondylia species caused galls on the plant specimens that had been dried and pressed. Examination of herbarium specimens has previously been used for assessment of distribution of the gall midge Rhopalomyia lawrenciae Kolesik causing inflation of leaves of Lawrencia squamata Nees in Lehm. (Malvaceae) in South Australian saltmarshes (Kolesik 1998) and Asphondylia floriformis and A. sarcocorniae infesting the samphire S. quinqueflora (Bunge ex Ung.– Stern.) A.J. Scott (Veenstra-Quah et al. 2007). The oldest plant specimen was collected from Port Lincoln in South Australia by J.S. Browne in 1874 (Fig. 6). Of the 243 specimens examined 45 had galls.

TAXON OMY AN D BIOL OGY Type species. Cecidomyia sarothamni Loew, 1850 Diagnosis. Asphondylia contains species that have ventrodistal spurs on the first tarsomeres, the ovipositor with a pair of large basal dorsal lobes, female flagellomeres 9–12 progressively shorter, the gonocoxite bearing a ventro-apical lobe and a dorsally situated gonostyle that is about as wide as long and bears two basally merged teeth. Asphondylia comprises nearly 300 described species worldwide (Gagné 2004), with 14 species known previously from Australia (Kolesik & VeenstraQuah 2008).

Asphondylia tecticorniae sp. n. Veenstra & Kolesik (Figs 2a,3a,4a,7–10, Table 3) Types. Victoria, Australia. Holotype male, Yaringa Marine National Park, Tyabb, Victoria (38°15′S, 145°14′E) emerged 8.ix.2007, A.A.V. reared from spherical leaf galls on T.

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Table 1 Herbarium specimens of Tecticornia arbuscula with galls caused by either Asphondylia tecticorniae or Asphondylia peelei held in Adelaide (AD) n = 13, Canberra (CANB) n = 4, Hobart (HO) n = 13, Melbourne (MEL) n = 5, New South Wales (NSW) n = 4 and Perth (PERTH) n = 6, with year of collection, collector and locality where specimens were collected Collection year 1874 1886 1919 1926 1929 1930 1930 1934 1937 1940 1959 1960 1968 1969 1974 1974 1974 1975 1975 1975 1976 1976 1977 1977 1979 1982 1982 1982 1983 1983 1983 1984 1984 1985 1985 1985 1986 1986 1990 1990 1992 1996 1996 2006 2007 2007

Collector

Locality

Hebarium ID

J.S. Browne S. Harris L. Rodway J.B. Cleland L. Rodway F.H. Long F.H. Long J. Marshall & A. Walkley A. Meebold R.A. Black D.E. Symon M.E. Phillips J.B. Cleland J. Carrick E.N.S. Jackson H. Posamentier K. Newbey D.E. Symon D.E. Symon D.E. Symon J.W. Parham J.W. Parham G. Jackson H.J. Eichler D.E. Symon A.C. Beauglehole & L.K.M. Elmore A.C. Beauglehole A.C. Beauglehole A.C. Beauglehole P. Wilson R.J. King & M. Wheeler N.N. Donner A.M. Buchanan B.M. Overton B.M. Overton A.M. Buchanan A.M. Buchanan A.M. Buchanan E. Mullins P. Collier K. Mills P.C. Heyligers A.M. Buchanan T. Jaques & T. Lewis P.A. Tyson A.M. Buchanan

Port Lincioln, SA Adelaide Bay, Tasmania George Bay, Tasmania American River, Kangaroo Island, SA East coast, Tasmania Ralph’s Bay, Tasmania Ralph’s Bay, Tasmania St.Kilda Beach, SA Hastings, Victoria Tooradin, Victoria Fowlers Bay, SA Ralph’s Bay, Tasmania Cape Spencer National Park, SA Port Gawler, SA Yorke Peninsula, SA Eurobodella, NSW Fitzgerald River National Park, WA Lower Coorong, SA South of Salt Creek, SA Lower Coorong, SA Double Creek, Tasmania Double Creek, Tasmania Kangaroo Island, SA Wapengo Lake, NSW Point Davenport, SA Quail Island, Victoria Breamlea Flora & Fauna Reserve, Victoria Rhyll Inlet Wildlife Reserve, Victoria Shallow Inlet, Victoria Rhyll Inlet Wildlife Reserve, Victoria North of Tarthra, NSW South-eastern region, SA Sedbury Creek, Tasmania Dudley Peninsula, Kangaroo Island, SA Dudley Peninsula, Kangaroo Island, SA Maclaines Creek, Tasmania Titan Point, Tasmania Hobart Airport, Tasmania Churchill Island, Victoria South Bruny Island, Tasmania South coast, NSW Wilsons Promontory, Victoria Pittwater Road, Tasmania Yorke Peninsula, SA Bakers Beach, Tasmania Bluff Road, Tasmania

MEL70556 HO125169 HO9991 AD97403186 AD97526034 HO300094 HO105781 AD98580519 NSW136565 MEL711332 AD98580518 CANB44995 AD97308528 AD96936096 AD9744418 NSW347427 PERTH2675773 AD98580522 PERTH2675836 PERTH2675943 HO8989 HO123774 AD96209191 CANB261325 PERTH2676044 MEL1564284 PERTH2675854 PERTH2675935 MEL105504 MEL1564281 NSW352606 AD98525444 HO408576 AD98623194 AD98623195 HO93807 HO123775 HO406444 CANB9015441 HO128049 NSW279880 CANB475049 HO316365 AD194491 HO545056 HO545401

arbuscula (R.Br.) K.A. Sheph. & Paul G. Wilson, collected 25.x.2007, SAMA 29-003367. Paratypes: 4 males, 5 females, 5 pupae, 4 larvae, SAMA 29-003368–29-003380. Other material: ANIC; same data as holotype but emerged 8–30.ix.2007. DNA COI analysis yielded identical sequences (GenBank accession numbers GQ379935, GQ379937, GQ379938, GQ379936) for four pupae collected in Yaringa Marine National Park, Tyabb, Victoria (38°15′S, 145°14′E) 25.xi.2006. Description. Male (Fig. 7a–g). Colour: antennae, thorax dorsally, sclerotised parts of abdomen dark brown, non-

sclerotised parts of abdomen yellowish orange; eyes dark brown. Wing length 3.5 mm (range 3.1–3.9, n = 4), subcostal cell weakly pigmented. Antenna: scape broadest distally, length 1.4 (1.3–1.6) ¥ breadth at distal end, 2.0 (1.4– 2.2) ¥ length pedicel, pedicel about as broad as long, first flagellomere 1.9 (1.7–2.2) ¥ length of scape. Flagellomeres evenly cylindrical, first flagellomere 5.0 (4.0–6.0) ¥ longer than wide, circumfila dense, equally distributed along segments. Eye facets close together, hexagonoid, eye bridge four facets long. Palpus three-segmented, length of third segment, as well as total length, variable. Frons with 9–10 setae per side. Claws of all legs similar in size and in shape, © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

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A A Veenstra et al.

(a)

(b)

horns and tip of posterior lower horn one-third distance between anterior horns and upper frontal horn. Prothoracic spiracle process short, and rounded. Abdominal dorsal spines simple, prominent pair on each side of last segment more or less straight. Larva (Fig. 9d). Colour: yellowish orange. Total length 4.0 mm (3.7–4.5, n = 4). Integument with dense spiculae. Head capsule pigmented. Spatula brown, with four pointed anterior teeth, outer teeth 2.9 times longer than two inner teeth, shaft narrow beyond midlength, abruptly widened posteriorly, one pair of setose lateral papillae adjacent to spatula.

Gall and biology (Figs 2a,3a,4a,5,6,10,17, Tables 1,3) Fig. 2. (a) Prothoracic spiracle on pupa of Asphondylia tecticorniae. (b) Prothoracic spiracle on pupa of Asphondylia peelei.

(a)

(b)

Fig. 3. (a) Gall on Tecticornia arbuscula caused by Asphondylia tecticorniae. (b) Gall on T. arbuscula caused by Asphondylia peelei. Scale bar = 1 mm.

slightly shorter than empodia. Genitalia: teeth on gonostyle merged into single, wide, lunate plate running along entire apical margin. Female (Fig. 8a–e). Wing length 3.4 mm (3.1–3.9, n = 5). Frons with 10–12 setae per side. First flagellomere 5.6 (3.8– 7.0) ¥ longer than wide, 12th flagellomere sometimes fused with 11th. Maxillary palpus three-segmented. Circumfila comprising two longitudinal bands connected by two transverse bands. Seventh abdominal sternite 1.5 ¥ length of sixth. Genitalia: ovipositor 3.7 ¥ length of seventh sternite, cerci setose. Other characters as in male. Pupa (Fig. 9a–c). Colour of mature pupa: abdomen orange, remaining parts pale brown. Total length 4.1 mm (3.5–4.4, n = 5). Antennal horns serrate mesally, 240–370 mm long. One upper and three lower frontal horns, four setose papillae between upper and lower horns. Lower frontal horns small, distance between base of anterior lower © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

Spherical leaf galls 5–11 mm in diameter (n = 20). A single larva develops in a chamber running perpendicular to the long axis of the gall (Fig. 4a). The inner wall of the chamber becomes covered with white fungal mycelium (Fig. 10a,c) which appears similar to that seen in A. floriformis and A. sarcocorniae (Veenstra-Quah et al. 2007). The mycelium becomes loosely attached to the plant cells (Fig. 10b) and in some places encircles the epidermal cells and enters the intercellular spaces becoming apoplastic. The location, as well as the orientation and number of chambers present in galls (Table 3) caused by A. tecticorniae and A. peelei differs. The length and width of galls caused by both new Asphondylia species (n = 20 for each) were measured using callipers. An independent t-test using the raw measurements of galls caused by each species indicated a significant difference in both the length (A. tecticorniae = 6–11 mm; A. peelei = 6–9 mm) (P < 0.05) and width (A. tecticorniae = 5–11 mm; A. peelei = 5–8 mm) (P < 0.05). Preliminary DNA analysis of the apoplastic mycelium associated with this genus also described by Rohfritsch (2008) and Veenstra-Quah et al. (2007) suggests that Botrysphaeria is present in galls induced by both A. tecticorniae and A. floriformis (T. Lebel pers. comm. 2010). Adair et al. (2009) found that Botryosphaeria dothidea (as its Dichomera synanamorph) was the most abundant and at times the only fungus present in galls induced by Asphondylia on Acacia species. Unlike galls caused by Asphondylia spp. on S. quinqueflora and those induced by A. peelei, the spherical galls of A. tecticorniae on T. arbuscula can be seen throughout the year but do not always contain a developing midge. Galls collected during September and October 2006 invariably contained either a developing midge or the parasitoid wasp Platygaster sp. 6 (Hymenoptera: Platygastridae). Geographical distribution overlaps with distribution range of T. arbuscula in coastal areas of southern New South Wales, Victoria, Tasmania, South Australia and south-east Western Australia. Etymology. The name of this species is derived from the generic name of the host plant.

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(b)

Fig. 4. (a) Cross-section through Asphondylia tecticorniae induced gall on Tecticornia arbuscula showing symmetrical shape of the gall and location of single chamber with pupa. (b) Crosssection through Asphondylia peeleiinduced gall showing asymmetrical shape of the gall and location of three chambers indicated – one with pupa and two each with single larva. Scale bar = 1 mm.

Fig. 5. Distribution of either Asphondylia tecticorniae or Asphondylia peelei in southern Australia based on herbarium specimens of Tecticornia arbuscula with galls.

Asphondylia peelei sp. n. Veenstra & Kolesik (Figs 2b,3b,11–13, Table 2) Types. Victoria, Australia. Holotype male, Yaringa Marine National Park, Tyabb (38°15′S, 145°14′E), Victoria emerged 6.ix.2008, A.A.V. reared from leaf galls collected from T. arbuscula (R.Br.) K.A. Sheph. & Paul G. Wilson, 3.ix.2008, SAMA 29-003381. Paratypes: 4 males, 3 females, 5 pupae, 4 larvae, SAMA 29-003382–SAMA 29-003392; ANIC; same data as holotype but emerged 12.ix–4.x.2008. DNA COI analysis yielded two sequences (GenBank accession numbers GQ379939 and GQ379940) for two larvae collected with holotype. Description. Male (Fig. 11a–h). Colour: antennae, thorax dorsally, sclerotised parts of abdomen dark brown, nonsclerotised parts of abdomen orange; eyes dark brown. Wing length 3.4 mm (range 3.1–3.5, n = 5), subcostal cell weakly pigmented. Antenna: scape broadest distally, length 1.5 (1.3– 2.0) ¥ breadth at distal end, 2.5 (2.0–3.0) ¥ length pedicel, pedicel about as broad as long, first flagellomere 1.8 (1.4– 2.3) ¥ length of scape. Flagellomeres evenly cylindrical, first

flagellomere 4.4 (3.6–5.0) ¥ longer than wide, circumfila dense, equally distributed along segments. Eye facets close together, hexagonoid, eye bridge four facets long. Maxillary palpus three-segmented, length of third segment, as well as total length, variable. Frons with 10–15 setae per side. Claws of all legs similar in size and in shape, slightly shorter than empodia. Genitalia: teeth on gonostyle merged into single, wide, lunate plate running along entire apical margin. Female (Fig. 12a–e). Wing length 3.7 mm (3.3–4.1, n = 5). Frons with 11–14 setae per side. First flagellomere 5.7 (4.8– 6.5) ¥ longer than wide, 12th flagellomere sometimes fused with 11th. Circumfila comprising two longitudinal bands connected by two transverse bands. Maxillary palpus threesegmented, length of third segment, as well as total length, variable. Seventh abdominal sternite 1.7 ¥ length of sixth. Genitalia: ovipositor 2.1 ¥ length of seventh sternite, cerci setose. Other characters as in male. Pupa (Fig. 13a–c). Colour of mature pupa: deep, dark brown. Total length 3.8 mm (3.5–4.2, n = 7). Antennal horns serrate mesally, 181–352 mm long. One upper and one lower © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

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Fig. 7. Asphondylia tecticorniae. Male: (a) head in frontal view; (b) maxillary palpus; (c) genitalia in dorsal view; (d) last three flagellomeres; (e) sixth flagellomere; (f) first tarsal segment; (g) last tarsal segment with claw and empodium.

Fig. 6. Herbarium specimen MEL70556 – Tecticornia arbuscula with galls caused by either Asphondylia tecticorniae or A. peelei. collected by J.S. Browne at Port Lincoln, South Australia in 1874.

frontal horn, with four setose papillae, setae of different lengths on either side of the lower horn. Prothoracic spiracle long and pointed. Abdominal dorsal spines simple, prominent pair on each side of last segment more or less straight. Larva (Fig. 13d). Colour: yellowish orange. Total length 3.1 mm (2.0–3.9, n = 11). Integument covered with dense spiculae. Head capsule pigmented. Spatula brown, with two pointed teeth of equal length, shaft narrow near middle, widened posteriorly, one pair of setose lateral papillae, setae of different lengths, adjacent to spatula.

Gall and biology (Figs 2b,3b,4b,5,6,17, Tables 1,3) Slightly asymmetrical leaf galls measuring 6–9 mm in length and 5–8 mm in width. A single larva or pupa is found in each chamber of a two- or three-chambered gall. Chambers run parallel to the long axis of the gall (Fig. 4b) and the inner wall of each chamber has white fungal mycelium present. Geographical distribution. As for A. tecticorniae – coastal areas of southern New South Wales, Victoria, Tasmania, South Australia and south-east Western Australia. © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

Fig. 8. Asphondylia tecticorniae. Female: (a) wing; (b) antenna; (c) sixth flagellomere; (d) basal lobes of ovipositor in dorsal view; (e) maxillary palpus.


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Fig. 9. Asphondylia sclerostegia. Pupa: (a) anterior part in ventral view with inset – enlarged view of antennal horns; (b) prothoracic spiracle; (c) last abdominal segment in dorsal view; larva: (d) first thoracic segment in ventral view.

(a)

(b)

Fig. 11. Asphondylia peelei. Male: (a) head in frontal view; (b) maxillary palpus; (c) genitalia in dorsal view; (d) gonostyle in posterior view; (e) last three flagellomeres; (f) sixth flagellomere; (g) first tarsal segment; (h) last tarsal segment with claw and empodium.

(c)

Fig. 10. (a) Cross-section through gall on Tecticornia arbuscula caused by Asphondylia tecticorniae with fungal mycelium (m) lined larval chamber (lc), containing a single pupa (p), gall cells (g) indicated. Cavity visible within pupa is a fixation artefact. (b) Mycelium on inner wall of chamber. (c) Longitudinal section through A. tecticorniae-induced gall with mycelium lined (m) chamber indicated.

Etymology. The species is named after Colleen Peele, who noticed a difference in shape while collecting galls for extraction and sequencing of fungal DNA from the mycelium lining galls induced by A. tecticorniae.

Comparative notes between new species

prothoracic spiracle that lacks a process in A. tecticorniae in contrast to A. peelei with a relatively long process. In larvae, the number of teeth on the spatula differ: four in A. tecticorniae and two in A. peelei. The results of an independent t-test indicated that there was a significant difference (P < 0.05, n = 5) between length of the ovipositor of A. tecticorniae (mean = 1.55 mm; 3.7 times the length of the seventh sternite) and A. peelei (mean = 1.05 mm; 2.1 times the length of the seventh sternite). Other differences between the two new species described and their Australian congeners infesting Chenopodiaceae are included in Table 3 and Figure 14 (Kolesik 1997; Veenstra-Quah et al. 2007; Kolesik & Veenstra-Quah 2008), Figure 15 (Kolesik 1997; Kolesik et al. 2005; Veenstra-Quah et al. 2007; Kolesik & Veenstra-Quah 2008), Figure 16 (Kolesik 1997; Veenstra-Quah et al. 2007; Kolesik & Veenstra-Quah 2008), Figure 17 (Veenstra-Quah et al. 2007), Table 2 (Kolesik 1997; Kolesik et al. 2005; Veenstra-Quah et al. 2007; Kolesik & Veenstra-Quah 2008) and Table 3 (Kolesik 1997; Veenstra-Quah et al. 2007; Kolesik & Veenstra-Quah 2008).

Morphological

Morphological differences between A. tecticorniae and A. peelei include the number of lower facial horns in pupa: three in A. tecticorniae and one in A. peelei, and the shape of the

DNA

Both the COI based tree (Fig. 15), and the tree based on a combination of morphological characters and sequences © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

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A A Veenstra et al. (Fig. 16) suggest a close relationship between A. tecticorniae and A. peelei. Stone and Schönrogge (2003) state that closely related gallers should cause structurally similar galls. This is indeed the case with A. tecticorniae and A. peelei where similar galls are induced on the same host plant T. arbuscula. COI analysis (Fig. 15) also confirmed the morpho-species with differences in 485 aligned nucleotides (Table 2) ranging from 39–40 nucleotides between A. sarcocorniae and A. vesicaria, and 41 nucleotides between A. tecticorniae sp. n. and A. peelei sp. n., to 188–189 nucleotides between A. floriformis and A. tecticorniae sp. n. and 192–193 nucleotides between A. mcneilli and A. floriformis. The intraspecific divergence was: 0 in A. tonsura and A. sarcocorniae; 0–1 in A. vesicaria, A. floriformis, A. tecticorniae sp. n. and A. peelei sp. n., and 6 in A. mcneilli. There was only a single usable sequence for both A. ericiformis and A. inflata so intraspecific variation could not be assessed for these species. Figure 16 showing one of two identical maximum parsimony trees recovered from a heuristic search of combined cytochrome c oxidase subunit I sequence data and the 10 morphological characters listed in Table 3, with A. vesicaria used as the outgroup. Two hundred and ninety-six of the 503 characters were found to be parsimony informative.

Fig. 12. Asphondylia peelei. Female: (a) wing; (b) antenna; (c) sixth flagellomere; (d) basal lobes of ovipositor in dorsal view; (e) maxillary palpus.

Fig. 13. Asphondylia peelei. Pupa: (a) anterior part in ventral view with inset – enlarged view of antennal horns; (b) prothoracic spiracle; (c) last abdominal segment in dorsal view; larva: (d) first thoracic segment in ventral view.

© 2011 The Authors Journal compilation © 2011 Australian Entomological Society

DIS CUS S ION Based on DNA analysis, insect morphology and gall form, two separate species of Asphondylia co-occur on T. arbuscula. Similar situations have been reported for a number of Asphondylia species, including Gagné and Waring (1990) who recorded 15 different species causing galls on various parts of the creosote bush (Larrea tridentata (Sessé & Mocino ex DC.)), two undescribed species galling different parts of ivy (Hedera rhombea (Miquel)) (Yukawa et al. 2003) and two different species on S. quinqueflora (Veenstra-Quah et al. 2007). In common with other Asphondylia groups there are morphological differences between these two new Asphondylia species and other Australian species previously described, namely A. ericiformis and A. inflata (Kolesik 1997), A. floriformis and A. sarcocorniae (Veenstra-Quah et al. 2007), and A. mcneilli, A. tonsura and A. vescaria (Kolesik & Veenstra-Quah 2008) (Fig. 14). In other descriptions of cecidomyiids, DNA analysis has also been used as a complementary taxonomic tool (Widenfalk et al. 2002;Yukawa et al. 2003; Kolesik et al. 2005; Sato & Yukawa 2006; Uechi & Yukawa 2006). Like A. sarcocorniae, A. peelei was not often found to be attacked by parasitoid wasps. The midges’ choice of oviposition site on the plant may be one of the reasons why these two species avoid wasp infestation (refer to Fig. 17 indicating putative oviposition sites). Once eggs are embedded in the plant tissue they are less accessible to the wasp, and the chemical cues that attract parasitoids described by Pérez-Maluf and Kaiser (1998) are reduced. Both S. quinqueflora and T. arbuscula have a gap between each developing vegetative ‘article’ or between each ‘bract’ if associated with a developing flower. A.

0.5 0.5 0.5 0.6 0.6 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.5 0.6 0.6 0.6 0.6 0.6 0.6 0.7 0.6

EF030746 EF030747 EF030748 EF030744 EF030745 EF030752 EF030753 EF030754 EF030749 EF030750 EF030751 DQ383812 DQ383811 GQ379935 GQ379937 GQ379938 GQ379936 GQ379939 GQ379940 EF030742 EF030743

0.5 0.5 0.5 0.6 0.6 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.5 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

*

AY278738 0.1

2

28

1

*

AY278712

3

* 0.0 0.0 0.3 0.3 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.3 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

146

153

4

0 * 0.0 0.3 0.3 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.3 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

146

153

5

1 1 * 0.3 0.3 0.1 0.1 0.1 0.2 0.2 0.2 0.2 0.3 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

146

153

6

96 96 95 * 0.0 0.4 0.4 0.4 0.4 0.4 0.4 0.4 0.5 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

174

172

7

8

151

156

9

151

156

10

151

156

11

146

152

12

146

152

13

147

151

96 39 39 39 69 69 70 96 39 39 39 69 69 70 95 40 40 40 70 70 71 0 103 103 103 122 122 121 * 103 103 103 122 122 121 0.4 * 0 0 75 75 76 0.4 0.0 * 0 75 75 76 0.4 0.0 0.0 * 75 75 76 0.4 0.3 0.3 0.3 * 0 1 0.4 0.3 0.3 0.3 0.0 * 1 0.4 0.3 0.3 0.3 0.0 0.0 * 0.4 0.2 0.2 0.2 0.4 0.4 0.4 0.5 0.4 0.4 0.4 0.4 0.4 0.4 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.7 0.7 0.7 0.6 0.6 0.6 0.6 0.6 0.6 0.6

174

172

GenBank accession number follows species name. Dasineura rubiformis was used as outgroup.

1 Dasineura rubiformis 2 Dasineura rubiformis 3 A. vesicaria 4 A. vesicaria 5 A. vesicaria 6 A. tonsura 7 A. tonsura 8 A. sarcocorniae 9 A. sarcocorniae 10 A. sarcocorniae 11 A. floriformis 12 A. floriformis 13 A. floriformis 14 A. inflata 15 A. ericiformis 16 A. sclerostegiae 17 A. sclerostegiae 18 A. sclerostegiae 19 A. sclerostegiae 20 A. peelei 21 A. peelei 22 A. mcneilli 23 A. mcneilli

Table 2 Nucleotide differences (above) and uncorrected divergences (below) 14

71 71 70 106 106 70 70 70 107 107 108 * 0.4 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6

162

165

15

101 101 102 140 140 112 112 112 127 127 128 130 * 0.5 0.5 0.5 0.5 0.5 0.5 0.6 0.6

156

161

16

179 179 178 173 173 175 175 175 188 188 189 174 158 * 0.0 0.0 0.0 0.1 0.1 0.3 0.4

187

187

17

179 179 178 173 173 175 175 175 188 188 189 174 158 0 * 0.0 0.0 0.1 0.1 0.3 0.4

187

187

18

179 179 178 173 173 175 175 175 188 188 189 174 158 0 0 * 0.0 0.1 0.1 0.3 0.4

187

187

19

179 179 178 173 173 175 175 175 188 188 189 174 158 0 0 0 * 0.1 0.1 0.3 0.4

187

187

20

165 165 164 175 175 168 168 168 186 186 187 162 143 41 41 41 41 * 0.0 0.3 0.4

181

181

21

165 165 164 175 175 168 168 168 186 186 187 162 143 41 41 41 41 0 * 0.3 0.4

181

181

22

171 171 171 182 182 175 175 175 192 192 193 170 163 83 83 83 83 85 85 * 0.2

192

188

23

168 168 168 174 174 167 167 167 182 182 183 169 164 120 120 120 120 124 124 6 *

187

183

New gall midges from Tecticornia 401


402

A A Veenstra et al.

Table 3 Chenopod host plants, structures affected by nine Australian Asphondylia gall midges and matrix of morphological characters Gall midge

A. vesicaria A. floriformis A. sarcocorniae A. tonsure A. inflate A. ericiformis A. tecticorniae A. peelei A. mcneilli

Host plant

Atriplex vesicaria Sarcocornia quinqueflora Sarcocornia quinqueflora Enchylaena tomentosa Tecticornia pergranulata ssp. elongata Tecticornia indica ssp. leiostachya Tecticornia arbuscula Tecticornia arbuscula Sclerolaena diacantha

Affected structure

G1

0 0 1 0 2 2 1 1 0

1 1 4 1 3 1 1 3 1

Character L1

L2

L3

P1

P2

A

M1

M2

M3

M4

5 2 2 5 2 3 2 2 5

1 0 1 1 0 0 0 0 1

1 1 0 1 0 0 1 0 0

0.7 0.2 0.1 0.7 0.6 0.5 0.0 0.5 0.7

3 3 3 1 1 0 3 1 1

2 2 1 1 1 1 1 1 1

0 2 1 0 2 1 1 0 0

0 1 1 0 1 0 0 1 0

6 3 5 4 5 5 6 5 5

2 0 1 2 1 2 2 2 2

Affected structure = flower (0) leaf (1) stem/branch (2). No. chambers/gall = single chamber (1) 2 to 3 (3) greater than 3 (4). Characters in larva (L), pupa (P), adults (A), male (M): G1 = number of chambers/gall 1 = 1, 3 = 2–3, 4 = 10–14+ L1 = number of lateral papillae L2 = spatula: inner teeth shorter (0) or as long as (1) outer teeth L3 = spatula: inner incision deeper than (1) or as deep as (0) outer incisions P1 = relative length of prothoracic spiracle process P2 = number of lower facial horns A = spur on first tarsal segment straight (0), bent at angle less than 90 deg (1) or more than 90 deg (2) M1 = teeth on gonostyle at angle smaller than 90 deg (0), 90 deg (1) or large than 90 deg (2) M2 = emargination between teeth on gonostyle V-shaped (0) or U-shaped (1) M3 = first flagellomere longer than wide by factor indicated M4 = palpi: segment 3 shorter than (0), as long as (1) or longer than (2) segment 2.

Fig. 14. Single tree recovered from maximum parsimony analysis of morphological characters of listed in Table 3. Tree length = 28, consistency index = 0.714, retention index = 0.619, homoplasy index = 0.286. Number above branch represents bootstrap value >50.

floriformis and A. tecticorniae have comparatively long ovipositors enabling them to deposit eggs in the narrow gap between articles or bracts, leaving the eggs more open to parasitoid attack. Hunt (1992) stated that wasp oviposition probably occurs in exposed gall midge eggs. By depositing eggs directly into the plant tissue A. sarcocorniae and A. peelei may effectively minimise the eggs exposure to wasp attack. This strategy is explained by Vinson (1998) who speculates that selective pressure from hymenopteran parasitoids forces host species to more effectively hide their eggs, and hosts that are plant borers are more likely to evade their attackers. However, Waring and Price (1989) concluded that it is unlikely that natural enemies such as parasitoids alone regulate host populations. Nor are they thought to influence gall morphology, because the relationship between the wasp and their host gall midge is direct, not influenced by the host plant © 2011 The Authors Journal compilation © 2011 Australian Entomological Society

Fig. 15. Cladogram of one of two identical maximum parsimony trees recovered from a heuristic search of cytochrome c oxidase subunit I sequence data from nine Australian Asphondylia species causing galls on chenopod plants. Dasineura rubiformis is the designated outgroup. Tree length = 1179, consistency index = 0.697, retention index = 0.853, homoplasy index = 0.321. Numbers above branches represent bootstrap values >50.


403

Fig. 16. Cladogram of one of two identical maximum parsimony trees recovered from a heuristic search of cytochrome c oxidase subunit I sequence data and morphological characters of listed in Table 3 from nine Australian Asphondylia species causing galls on chenopod plants. Asphondylia vesicaria was used as outgroup. Tree length = 844, consistency index = 0.779, retention index = 0.673, homoplasy index = 0.220. Numbers above branches represent bootstrap values >50. L, larval spatula; P, pupal prothoracic spiracle.

evolution of enhanced protection strategies such as depositing eggs directly into the host plant tissue. Actual observation of the behaviour of the four Asphondylia species in question is required to confirm or reject the putative location of the oviposition sites suggested above.

ACK N OW L EDGEMEN T S

Fig. 17. Generalised chenopod vegetative articles (modified leaves) and bracts (site of inflorescence) with putative location of oviposition sites for Asphondylia peelei, A. tecticorniae, A. floriformis and A. sarcocorniae indicated.

(Skuhravá & Thuróczy 2007). Darrouzet-Nardi et al. (2006) stated that hymenopteran parasitoids do suppress populations of host insects in many terrestrial ecosystems. Consequently, a high level of infestation by parasitoids such as Platygaster sp. 6 may reduce the reproductive success of their respective hosts – A. tecticorniae and A. floriformis. Joy and Crespi (2007) suggest that speciation can be associated with shifting to different parts of the same host plant and increased rates of change in ecologically important traits such as ovipositor length. Additionally, Stone and Schönrogge (2003) suggest that selection pressure imposed by enemies – in this case parasitoid wasps – is a probable adaptive explanation for the

We thank Leigh Ackland, Guang Shi, Loveleen Kumar, David Freestone (Deakin University), Teresa Lebel and Alison Vaughan (Royal Botanic Gardens Melbourne), for their support, encouragement and expertise; Kelly Shepherd (Western Australian Herbarium, Perth) who confirmed identification of the host plant species and advised on chenopod morphology, Matthew Buffington (Systematic Entomology Laboratory, US Department of Agriculture/Department of Entomology, Smithsonian Institution, Washingston) who identified the parasitoid wasp, Mick Douglas and Aaron Ledden (Parks Victoria) for their fieldwork assistance, Cuong Huynh (Deakin University) for mounting midge specimens, Bruce Abaloz (University of Melbourne) for gall histology preparations, Josephine and Steve Milne, and Colleen Peele for their help in gall collection. We acknowledge Parks Victoria for kindly permitting collection in the Yaringa Marine National Park, and the directors and staff of AD, CANB, HO, MEL, PERTH and NSW for allowing access to herbarium collections. We thank Raymond Gagné (Systematic Entomology Laboratory, USDA, Washington DC) for reviewing an early draft of the paper. This work was supported by a Deakin University, Faculty of Science and Technology start-up grant to A.A.V.

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